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Spontaneous mutations in the galactose operons of Streptomyces coelicolor A3 (2) and Streptomyces lividans 66.

Mutations can be divided into two classes: point mutations (base changes, frame shifts) and DNA rearrangements (deletions, insertions, inversions etc.). In Escherichia coli K 12, DNA insertions account for up to 40% of spontaneous mutations that inactivate genes (for review, see CULLUM 1985) and the Insertion Sequences involved can also mediate deletions and inversions. We started to study spontaneous mutations in Streptomyces in the expectation of isolating transposable elements and chose the galactose genes as convenient system. When selection is made for resistance to the galactose analogue 2-deoxygalactose (2dgalR), gal K mutants are obtained that are defective in the enzyme galactokinase (Kendall et al. 1987). We isolated spontaneous 2dgalR mutations in the closely related strain S. coelicolor A 3 (2) and S. lividans 66 and cloned the gal K gene of the former strain. Adams et al. (1988) have cloned and sequenced the gal genes of S. lividans 66 and shown that they form an operon, the restriction map appears to be identical to that of S. coelicolor A 3 (2). In this paper we describe the characterisation of spontaneous 2 dgalR mutants in S. coelicolor A 3 (2) and S. lividans 66.

Chromosome Deletion

Structural complexity and mechanistic diversity of MECOM rearrangements in myeloid neoplasms.

Rearrangements involving MECOM at chromosome 3q26.2 are recurrent in myeloid neoplasms, classically represented by inv(3)(q21q26.2) and t(3;3)(q21;q26.2), which reposition the GATA2-distal haematopoietic enhancer and drive aberrant EVI1 overexpression. However, the full structural and mechanistic diversity of MECOM rearrangements (MECOM-r) is yet to be explored. We retrospectively analysed 97 cases with cytogenetically defined MECOM-r and identified 12 with complex rearrangements using GTG-banded karyotyping and tri-colour interphase/metaphase fluorescence in situ hybridisation analyses. These 12 cases demonstrated remarkable structural heterogeneity. The abnormalities encompassed translocations, inversions, insertions, duplications, and deletions, which often coexisted within the same specimen as multiple rearranged subclones. Insertional events emerged as a distinct mechanism of MECOM activation. These encompassed insertions of MYNN and/or MECOM into chromosomes 1 and 6, insertion of chromosome 8 segment into MECOM, and inverted insertions between homologous chromosome 3 segments. Recurrent breakpoints at 3q21 across multiple cases, together with localised copy number imbalances frequently involving the MYNN and GOLIM4 loci at 3q26.2, underscore the architectural fragility of these two regions. Co-occurring abnormalities such as -5/del(5q), -7/del(7q), and TP53 loss were common, reflecting a permissive genomic background for chromosomal reassembly. Our findings expand the mechanistic landscape of MECOM-r beyond canonical inv(3)/t(3;3), establishing 3q21 and 3q26.2 as structural 'hotspots' and genomic instability hubs. Distinct from fusion-driven oncogenes such as KMT2A, MECOM activation results from enhancer hijacking and regional structural remodelling, leading to EVI1 overexpression and clonal evolution in myeloid malignancies.

Humans

Cytogenetic analysis of 57 primary prostatic adenocarcinomas.

Cytogenetic analysis after short-term culture in vitro of primary tumor samples was attempted in 82 patients with prostatic cancer. Tumor material was obtained by radical prostatectomy or transurethral resection. Successful cytogenetic studies were performed on 57 tumors of which five were well, 30 moderately, and 22 poorly differentiated adenocarcinomas. Only normal karyotypes were found in 24 tumors. Structural nonclonal aberrations were detected in 18 and clonal karyotypic abnormalities in 15 tumors. The most common clonal numerical aberration was loss of the Y chromosome; a missing Y was found in six tumors, in three of these as the sole anomaly. Clonal structural chromosomal rearrangements, usually accompanied by numerical changes, were detected in 12 tumors. The rearrangements involved 18 of the 22 autosomes and the X chromosome. Chromosomes 1, 7, and 10 were most frequently affected. Deletions, duplications, inversions, insertions, and balanced as well as unbalanced translocations were represented. The breakpoints in chromosome 1 were scattered along both the short and long arms with no obvious clustering, whereas those in chromosomes 7 and 10 were clustered at bands 7q22 (two deletions and two duplications in four different tumors) and 10q24 (two translocations, one deletion, and one inversion in four tumors). One additional tumor displayed a derivative chromosome 10 with a breakpoint in 10q23, and one had monosomy 10. Altogether, these abnormalities resulted in loss of 10q24----qter in five tumors. Monosomy 8 and rearrangements of the short arm of chromosome 8 leading to loss of 8p21----pter were seen in four tumors. Double minute chromosomes were found in two tumors.

Adenocarcinoma

SfiI genomic cleavage map of Escherichia coli K-12 strain MG1655.

An SfiI restriction map of Escherichia coli K-12 strain MG1655 is presented. The map contains thirty-one cleavage sites separating fragments ranging in size from 407 kb to 3.7 kb. Several techniques were used in the construction of this map, including CHEF pulsed field gel electrophoresis; physical analysis of a set of twenty-six auxotrophic transposon insertions; correlation with the restriction map of Kohara and coworkers using the commercially available E. coli Gene Mapping Membranes; analysis of publicly available sequence information; and correlation of the above data with the combined genetic and physical map developed by Rudd, et al. The combination of these techniques has yielded a map in which all but one site can be localized within a range of +/- 2 kb, and over half the sites can be localized precisely by sequence data. Two sites present in the EcoSeq5 sequence database are not cleaved in MG1655 and four sites are noted to be sensitive to methylation by the dcm methylase. This map, combined with the NotI physical map of MG1655, can aid in the rapid, precise mapping of several different types of genetic alterations, including transposon mediated mutations and other insertions, inversions, deletions and duplications.

Base Sequence

Genetic relationship of two highly studied Synechococcus strains designated Anacystis nidulans.

The cyanobacteria Synechococcus sp. strain PCC 7942 and Synechococcus sp. strain PCC 6301 are very closely related and both have been designated by the binomial Anacystis nidulans. The only established difference between the two strains is the superior transformation properties of strain PCC 7942. Significant homology between the rRNA genes of these strains was demonstrated by the ability of an rRNA operon from strain PCC 6301, interrupted by a spectinomycin and streptomycin resistance marker, to transform strain PCC 7942 by recombining with and replacing an endogenous rRNA operon. Restriction fragment length polymorphism data indicated that the chromosomes of the two strains were conserved around the three psbA loci, the two rRNA operons, and the psbDI locus. However, multiple polymorphisms were detected downstream of the psbDII locus, identifying a DNA rearrangement such as an inversion, insertion, or deletion within the chromosome. Analysis of genome structure by pulsed-field gel electrophoresis of large NotI restriction fragments showed only two bands that were visibly shifted between the chromosomes of the two strains. These data support their very close genetic relationship and the feasibility of studying genes derived from strain PCC 6301 in the highly transformable PCC 7942 strain.

Blotting, Southern

Report of the committee on structural chromosome changes in neoplasia.

An enormous amount of data on neoplasia-associated chromosome aberrations has accumulated over the past two years. More than 4,000 tumors with a chromosome anomaly identified by banding have been published since HGM9, and the total number of cases contained in the registry on which the Catalog of Chromosome Aberrations in Cancer (Mitelman, 1988) is based is now well above 12,000. The information presently available is, however, still in many respects incomplete. First, the data is heavily biased in favor of hematologic disorders. Solid tumors comprise only 20% of the total data base, which is totally disproportionate to the relative contribution of these disorders to human cancer morbidity and mortality. For example, malignant epithelial tumors (carcinomas), which cause almost 80% of all cancer deaths in man, comprise only 7% of the total. Second, our knowledge about early stage tumors is very limited. For example, the great majority of the solid tumors that have been studied cytogenetically have been metastatic lesions or effusions (advanced tumors usually have a large number of complex structural and numerical chromosome aberrations). Obviously, many more such neoplasms will have to be studied before the primary (pathogenetically essential) changes can be distinguished from the confusing variety of secondary abnormalities that may dominate the karyotype. It should be noted that secondary changes may also be nonrandom, and may be important for tumor progression. Therefore, no attempt has been made in this report to distinguish between primary and secondary changes. All nonrandomly occurring abnormalities that met the criteria for inclusion are listed in Table 1 irrespective of their presumed pathogenetic significance. Results of molecular genetic studies (e.g. the demonstration of loss of heterozygosity or gene amplification) were not considered, although they may be included in the HGM10.5 report. A total of 149 nonrandom chromosome changes were identified (Table 1) in 43 different types of neoplastic disorders, including hematologic diseases and malignant lymphomas, as well as tumors of epithelial, mesenchymal, neurogenic, germ cell, and melanocytic origin. The aberrations comprise a variety of structural chromosome rearrangements (translocations, inversions, insertions, deletions, duplications and isochromosomes), and all chromosomes, except the Y chromosome are involved. The great majority (121 of the 149 identified aberrations) represent well-defined, specific structural changes. More than half of them are consistently associated with a particular morphologic disease characteristic. Twenty-eight of the aberrations, although nonrandom, are not characterized as well. Most are deletions or translocations that only affect a certain chromosome region, often spanning several bands.(ABSTRACT TRUNCATED AT 400 WORDS)

Chromosome Aberrations

Spectrum of mutations produced by specific types of restriction enzyme-induced double-strand breaks.

Rejoining of DNA double-strand breaks (DSB) plays a central role in the various processes leading to DNA rearrangements. We have analyzed DNA alterations induced by restriction enzymes that produce DSB with specific types of ends. Restriction enzymes were electroporated into a human lymphoblastoid cell line that stably maintains pHAZE, an EBV-based vector containing the lacZ gene. After allowing time for DSB repair, pHAZE DNA was rescued and screened in Escherichia coli. Mapping and sequence analysis of mutant copies of pHAZE indicated that restriction enzymes induced all classes of alterations except base substitutions (base deletions and insertions, large-scale deletions, inversions, and insertions). The spectra of alterations were distinctive for each enzyme and appear to be the consequence of specific end-modification processes.

Base Sequence

Hybrid dysgenesis-induced revertants of insertions at the 5' end of the rudimentary gene in Drosophila melanogaster: transposon-induced control mutations.

Mutations in the 5' control region of the rudimentary (r) gene of Drosophila melanogaster were generated by using hybrid dysgenesis to mobilize P elements that were already inserted within this region. Eighteen new mutations out of 7793 chromosomes were isolated. Among the mutations were small insertions, deletions and inversions. All five of the deletions deleted into the 5' coding region of the r gene resulting in a severe mutant phenotype. The inversions and small insertions left the coding region intact, but altered the 5' control region. Northern analyses of the message levels in adult females indicated that these mutations alter the amount of the wild-type rudimentary message. These data also indicated that there is a region about 800 bp upstream of the start of transcription which is necessary for normal r expression. This region is not involved with the temporal and spatial regulation of r gene expression, but with the quantitative expression. An analysis of the DNA changes associated with each of the mutations demonstrates that P elements usually mobilize in an imprecise manner. Of the 22 mutations that have been isolated to date, only three are precise excisions. This means that P elements are potent mutators of the genome.

Animals

Bacteriophage P1 derivatives unaffected in their growth by a large inversion or by IS insertions at various locations.

Several plaque-forming phage P1 derivatives carrying DNA rearrangements associated with IS elements are described. They have IS1, IS3 and IS5 inserted in four distinct locations, all of which are non-essential regions for phage P1 propagation. One derivative carries a genome segment, inverted relative to the one in the P1 wild-type genome, between two inverted copies of IS1. The inverted DNA segment spans about 23 kb of the 90 kb long P1 genome and it includes the invertible C segment. This phage is as viable as an isomeric P1 which carries the relevant segment in its original orientation. These results are discussed with regard to the genome organization of phage P1.

Coliphages

Segregation of an insertional chromosome rearrangement in 3 generations.

The interstitial deletion of a segment of chromosome 13, 13q21 leads to 13q22, and its inversion and insertion into the long arm of chromosome 3 at breakpoint q12, was found to segregate in 3 generations of a family. Segregation of this 3 break rearrangement gave rise to individuals monosomic, trisomic, or balanced for the involved segment. Monosomy for 13q21 leads to 13q22 was associated with mental retardation, expressive aphasia, microcephaly, hand abnormalities, and short stature. Partially trisomic individuals had normal mentality, extremely high arched palate, and mild dysmorphic features. There was no evidence for retinoblastoma in the individuals examined. The balanced carriers were normal. Comparison of monosomic individuals with one previous report of a similar deletion reveals marked phenotypic similarities.

Adult

Structure of the gas vesicle plasmid in Halobacterium halobium: inversion isomers, inverted repeats, and insertion sequences.

Halobacterium-halobium NRC-1 harbors a 200-kb plasmid, pNRC100, which contains a cluster of genes for synthesis of buoyant gas-filled vesicles. Physical mapping of pNRC100 by using pulsed-field gel electrophoresis showed the presence of a large (35 to 38-kb) inverted repeat (IR) sequence. Inversion isomers of pNRC100 were demonstrated by Southern hybridization analysis using two restriction enzymes, AflII and SfiI, that cut asymmetrically within the intervening small single-copy region and the large single-copy region, respectively, but not within the large IRs. No inversion isomers were observed for a deletion derivative of pNRC100 lacking one IR, which suggests that both copies are required for inversion to occur. Additionally, the identities and approximate positions of 17 insertion sequences (IS) in pNRC100 were determined by Southern hybridization and limited nucleotide sequence analysis across the IS element-target site junctions: ISH2, a 0.5-kb element, was found in four copies; ISH3, a 1.4-kb heterogeneous family of elements, was present in seven copies; ISH8, a 1.4-kb element, was found in five copies; and ISH50, a 1.0-kb element, was present in a single copy. The large IRs terminated at an ISH2 element at one end and an ISH3 element at the other end. pNRC100 is similar in structure to chloroplast and mitochondrial genomes, which contain large IRs and other large halobacterial and prokaryotic plasmids that are reservoirs of IS elements but lack the large IRs.

Base Sequence

Frequent occurrence of cytogenetic abnormalities in sporadic nonmedullary thyroid carcinoma.

Cytogenetic studies may provide important clues to the molecular pathogenesis of thyroid neoplasia. Thus, the authors attempted cytogenetic studies on 12 thyroid carcinomas: seven papillary, three follicular, and two anaplastic. Successful cytogenetic results were obtained on all 12 tumors; nine (75%) had one or more chromosomally abnormal clones. Four of the papillary carcinomas had a simple clonal karyotype, and three had no apparent chromosome abnormality. All four abnormal papillary tumors contained an anomaly of a chromosome 10q arm. In one instance, an inv(10)(q11.2q21.2) was observed in a Grade 2 papillary carcinoma as the sole acquired abnormality. In another case, an inversion or insertion involving 10q21.2 was found in a Grade 1 papillary tumor. The karyotype of a third tumor, a Grade 1 papillary carcinoma, was 46,XX,der(5)t(5;10)(p15.3;q11),der(9)t(9;?)(q11;?). A fourth abnormal papillary carcinoma, a Grade 1 tumor, had a t(6;10)(q21;q26.1) as the sole abnormality. Each of the five follicular or anaplastic carcinomas had a complex clonal karyotype. The three follicular carcinomas contained an abnormality of 3p25-p21, along with several other chromosome abnormalities.

Adenocarcinoma

Identification of an enhancer-like element upstream from a cell cycle dependent human H4 histone gene.

We have identified a segment of DNA in the region 6,500 nucleotides upstream from a cell-cycle-dependent human H4 histone gene (pF0108A) which exhibits properties of an enhancer element. This distal element is not required for cap site initiation from the F0108A H4 histone gene. When the enhancer element is present in the genome as a stable integrated sequence, either in its natural upstream location or in a construct where the element is moved just upstream from the proximal promoter sequences, a 25-fold increase in the level of human H4 histone RNAs is observed. This increased level of mRNA reflects an increase in the rate of transcription. The enhancer effect is also observed when the distal element is inserted in inverse orientation with respect to this gene. In addition, the far upstream element can increase expression of a prokaryotic chloramphenicol acetyl transferase (CAT) gene under control of the simian virus 40 (SV40) early promotor, indicating that the ability to influence transcription is not confined to the gene with which it is normally associated. The ability of the histone gene distal enhancer element to function in both mouse and human cells indicates that transacting regulatory factors encoded by either the human or murine genome are capable of mediating the functional properties of this element, further supporting the cross-species compatibility of regulatory sequences and molecules that influence transcription of human histone genes.

Acetyltransferases

Sister chromatid exchange and chromosome organization based on a bromodeoxyuridine Giemsa-C-banding technique (TC-banding).

Hoechst 33258 fluorescent staining of bromodeoxyuridine substituted chromosomes provided a high resolution technique for following the segregation of replicated chromosomal DNA (Latt, 1973). Modifications have produced the same results after Giemsa staining (Wolff and Perry, 1975). Since this does not necessarily require Hoechst (Korenberg and Freedlander, 1975), we call this bromodeoxyuridine-Giemsa banding (BG-banding). We here describe a further modification which allows one to follow the T-rich strand of the AT-rich satellite DNA of C-band heterochromatin. We call this TC-banding. This technique was used to examine metacentric marker chromosomes found in mouse L-cells that contain many interstitial blocks of centromeric-type heterochromatin in each arm plus the usual two blocks of centromeric heterochromatin. One of the advantages of this technique for such chromosomes is that it is possible to distinguish first from second cell cycle sister chromatid exchange and unambiguously detect centromeric sister chromatid exchange. We found some chromosomes to have high rates of centromeric sister chromatid exchange. After one cycle in bromodeoxyuridine we could examine the satellite polarity of the heterochromatic DNA. Since there was no change in satellite polarity in any of the heterochromatic blocks, marker chromosomes could not have been formed by paracentric inversions, inverted insertions or inverted translocations. These results allow the formulation of several rules of chromosome organization.

Animals

Characterization of mutations induced by 2-(N-acetoxy-N-acetyl)aminofluorene in the dihydrofolate reductase gene of cultured hamster cells.

To determine the types of alterations in gene structure that are induced by the carcinogen 2-(N-acetoxy-N-acetyl)aminofluorene, we used this compound to generate mutations at the dihydrofolate reductase (DHFR) locus (DHFR) in Chinese hamster ovary cells. Twenty-nine independent enzyme-deficient mutants were isolated. A profile of the 26-kilobase (kb)-long gene was obtained by Southern blot analysis of the mutant and parental DNAs digested with BstEII/Kpn I. Hybridization to a mixed probe of 10 DHFR genomic and cDNA fragments revealed 12 bands that scan 34 kb. Twenty-one DHFR- clones (72%) contained small mutations (changes less than 100 base pairs in size). Large or small deletions involving various parts of the gene occurred in eight of the mutants (28%). A large deletion (greater than 35 kb) with 5' and 3' breakpoints mapping to approximately the same location was noted in four mutants. One mutant has undergone a deletion of 550-900 bp that eliminated the first coding exon. Concomitantly, a chromosomal event (either translocation, insertion, or inversion) has separated the 5' flank from the body of the gene. In another mutant, four deletions have occurred at the DHFR 5' end and internally. Restriction fragment length polymorphism analysis of the mutant DNAs with exon-specific probes localized three mutations. One mutant has lost a Taq I (TCGA) site, and another has lost a Sac I (GAGCTC) site. In a third, a GC----TA transversion has created a BstEII (GGTNACC) site. Finally, we used HPLC to determine the ratio of acetylated (12%) to deacetylated (88%) 2-aminofluorene adducts formed in the parental cells. A correlation between the mutational specificities and the conformational changes induced by the two types of DNA adducts is discussed.

2-Acetylaminofluorene

A somatic gene rearrangement contributing to genetic diversity in maize.

We have discovered a somatic genomic rearrangement that occurs at high frequency at a duplicated zein locus in certain cultures of the maize inbred line A188. The rearranged allele arises from the duplication by a two-step process involving a homologous recombination and a second event, which may be a deletion, inversion, or insertion; both steps always occur together. The frequency of rearrangement is lower in homozygous states of the parental allele than in heterozygotes. In both cases, the rearrangement is shown to be mitotic. The rearranged product can be transmitted through meiosis, providing another mechanism for genome evolution in higher eukaryotes.

Base Sequence

Gene order comparisons for phylogenetic inference: evolution of the mitochondrial genome.

Detailed knowledge of gene maps or even complete nucleotide sequences for small genomes leads to the feasibility of evolutionary inference based on the macrostructure of entire genomes, rather than on the traditional comparison of homologous versions of a single gene in different organisms. The mathematical modeling of evolution at the genomic level, however, and the associated inferential apparatus are qualitatively different from the usual sequence comparison theory developed to study evolution at the level of individual gene sequences. We describe the construction of a database of 16 mitochondrial gene orders from fungi and other eukaryotes by using complete or nearly complete genomic sequences; propose a measure of gene order rearrangement based on the minimal set of chromosomal inversions, transpositions, insertions, and deletions necessary to convert the order in one genome to that of the other; report on algorithm design and the development of the DERANGE software for the calculation of this measure; and present the results of analyzing the mitochondrial data with the aid of this tool.

Biological Evolution